Estrogen stimulates SREBP2 expression in hepatic cell lines via an estrogen response element in the SREBP2 promoterSpringer Science and Business Media LLC - - 2019
Yahan Meng, Lu Zong
AbstractObjectiveHypoestrogenism in women is strongly associated with menopause and it can lead to lipid disorder, which predisposes people to premature cardiovascular disease. However, the mechanism of lipid disorder remains unclear. Sterol regulatory element-binding protein 2 (SREBP2) is the key transcription factor regulating cholesterol metabolism. We hypothesize that estrogen regulates SREBP2 transcription through an estrogen response element (ERE) in the SREBP2 promoter region.
MethodsHuman hepatoblastoma cells (HepG2) were treated with dose-dependent concentrations of estradiol (E2) for 24 h. Then, SREBP2 expression was determined via real-time PCR and immunofluorescence. The expressions of the SREBP2 downstream target genes HMGCR and LDLR were determined via real-time PCR. Lipid secretion in the culture media of HepG2 cells was measured using ELISA. Through bioinformatics analysis, we identified high-scoring ERE-like sequences in the SREBP2 gene promoter. Chromatin immunoprecipitation analysis was used to confirm the ERE. DNA fragments of the putative or mutated ERE-like sequence were synthesized and ligated into pGL3-basic plasmid to construct the SREBP2 promoter luciferase reporter systems. SREBP2-Luciferase (SREBP2-Luc), SREBP2-Mutation (SREBP2-Mut) and the blank control were transfected into hepatic cell lines. Luciferase activities were measured using the dual-luciferase reporter assay system. Chromatin immunoprecipitation analysis and the luciferase reporter assay were repeated in human hepatoma cells (HuH-7).
ResultsWe found that E2dose-dependently increased the expression of SREBP2 in HepG2 cells and that the increased levels were blocked when treated with an estrogen receptor-alpha antagonist. Additionally, E2increased both HMGCR and LDLR expression and lipid secretion in HepG2 cells. Notably, we identified a functional ERE in the SREBP2 gene promoter, to which E2could specifically bind and induce transcription.
ConclusionsAn ERE was identified in the SREBP2 gene promoter. It mediates the regulation of SREBP2 expression by estrogen in hepatocytes. This study provides a mechanism to link cardiovascular disease with estrogen.
Red blood cell shape and deformability in the context of the functional evolution of its membrane structureSpringer Science and Business Media LLC - Tập 17 - Trang 171-181 - 2012
Saša Svetina
It is proposed that it is possible to identify some of the problems that had to be solved in the course of evolution for the red blood cell (RBC) to achieve its present day effectiveness, by studying the behavior of systems featuring different, partial characteristics of its membrane. The appropriateness of the RBC volume to membrane area ratio for its circulation in the blood is interpreted on the basis of an analysis of the shape behavior of phospholipid vesicles. The role of the membrane skeleton is associated with preventing an RBC from transforming into a budded shape, which could form in its absence due to curvature-dependent transmembrane protein-membrane interaction. It is shown that, by causing the formation of echinocytes, the skeleton also acts protectively when, in vesicles with a bilayer membrane, the budded shapes would form due to increasing difference between the areas of their outer and inner layers.
Cloning and expression of a new inositol 1,4,5-trisphosphate receptor type 1 splice variant in adult rat atrial myocytesSpringer Science and Business Media LLC - Tập 17 Số 1 - 2012
Krishna Subedi, Thoudam Debraj Singh, Joon-Chul Kim, Seung Hyo Woo
AbstractInositol 1,4,5-trisphosphate receptor type 1 (IP3R1) is already known to be highly expressed in the brain, and is found in many other tissues, including the atrium of the heart. Although the complete primary structure of IP3R1 in the rat brain has been reported, the complete sequence of an IP3R1 clone from atrial myocytes has not been reported. We isolated an IP3R1 complementary DNA (cDNA) clone from isolated adult rat atrial myocytes, and found a new splice variant of IP3R1 that was different from a previously reported IP3R1 cDNA clone obtained from a rat brain (NCBI GenBank accession number: NM_001007235). Our clone had 99% similarity with the rat brain IP3R1 sequence; the exceptions were 39 amino acid deletions at the position of 1693–1731, and the deletion of phenylalanine at position 1372 that lay in the regulatory region. Compared with the rat brain IP3R1, in our clone proline was replaced with serine at residue 2439, and alanine was substituted for valine at residue 2445. These changes lie adjacent to or within the fifth transmembrane domain (2440–2462). Although such changes in the amino acid sequences were different from the rat brain IP3R1 clone, they were conserved in human or mouse IP3R1. We produced a plasmid construct expressing the atrial IP3R1 together with green fluorescent protein (GFP), and successfully overexpressed the atrial IP3R1 in the adult atrial cell line HL-1. Further investigation is needed on the physiological significance of the new splice variant in atrial cell function.
The transcript expression profile of the leptin receptor-coding gene assayed with the oligonucleotide microarray technique — Could this be an Anorexia nervosa marker?Springer Science and Business Media LLC - Tập 11 - Trang 62-69 - 2006
Małgorzata Janas-Kozik, Urszula Mazurek, Irena Krupka-Matuszczyk, Małgorzata Stachowicz, Joanna Głogowska-Ligus, Tadeusz Wilczok
Anorexia nervosa is a serious eating disorder with the highest mortality rate of any psychiatric disorder. The DSM-IV classification differentiates two AN types: the restricting type (AN-R) and the binge-eating/purging type (AN-BP). Leptin (LEP) levels can be thought of as a signal to the body of its energy reserves. The leptin receptor (including all its mRNA isoforms) is expressed in many tissues. Our aim was to discover the transcript expression profile of the LEP receptor-coding gene in the peripheral blood mononuclears in AN-R and AN-BP patients. Three young women suffering from Anorexia nervosa (one with AN-BP and two with AN-R) took part in the study, along with three non-anorexic subjects as our reference group. LEP receptor gene expression was examined using the oligonucleotide microarray method (HG-U133A, Affymetrix). The results were normalized using RMAExpress. Next, the accumulation analysis method was used (clustering). Hierarchical clustering resulted in three groups of separate clusters. The first group (cluster I) consisted of AN-R patients. The next group (cluster II) consisted of reference group patients suffering from different psychic disorders not related to eating disorders. Cluster III consisted of two patients — the first with AN-BP and the second with an adaptive disorder.
The proliferation and differentiation of osteoblasts in co-culture with human umbilical vein endothelial cells: An improved analysis using fluorescence-activated cell sortingSpringer Science and Business Media LLC - - 2010
Yu Zhang, Andreas Schedle, M Matĕjka, Xiaohui Rausch‐Fan, Oleh Andrukhov
AbstractThe interaction of osteoblasts and endothelial cells plays a pivotal role in osteogenesis. This interaction has been extensively studied using their direct co-culture in vitro. However, co-culture experiments require clear discrimination between the two different cell types in the mixture, but this was rarely achieved. This study is the first to use fluorescence-activated cell sorting (FACS) for the separation and quantitative analysis of the proliferation and differentiation of MG-63 cells grown in direct co-culture with human umbilical vein endothelial cells (HUVECs). The cells of the MG-63 cell line have properties consistent with the characteristics of normal osteoblasts. We labeled HUVECs with fluorescent antibody against CD31 and used FACS to measure the proportions of each cell type and to separate them based on their different fluorescence intensities. The rate of proliferation of the MG-63 cells was estimated based on a count of the total viable cells and the proportion of MG-63 cells in the mixture. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), collagen type 1 (Coll-1) and osteocalcin (OC) in the MG-63 cells were measured via real-time PCR after the separation via FACS. We found that HUVECs stimulated the proliferation of the MG-63 cells after 72 h of co-culture, and inhibited it after 120 h of co-culture. The mRNA expression levels of ALP and Coll-1 significantly increased, whereas that of OC significantly decreased in MG-63 after co-culture with HUVECs. Using FACS for the quantitative analysis of the proliferation and differentiation of osteoblasts directly interacting with endothelial cells could have merit for further co-culture research.
Obesity-induced downregulation of miR-192 exacerbates lipopolysaccharide-induced acute lung injury by promoting macrophage activationSpringer Science and Business Media LLC - - 2024
Siqi Wu, Wenjing Tang, Ling Liu, Ke Wei, Yin Tang, Jingyue Ma, Hongbin Li, Yichan Ao
Macrophage activation may play a crucial role in the increased susceptibility of obese individuals to acute lung injury (ALI). Dysregulation of miRNA, which is involved in various inflammatory diseases, is often observed in obesity. This study aimed to investigate the role of miR-192 in lipopolysaccharide (LPS)-induced ALI in obese mice and its mechanism of dysregulation in obesity. Human lung tissues were obtained from obese patients (BMI ≥ 30.0 kg/m2) and control patients (BMI 18.5–24.9 kg/m2). An obese mouse model was established by feeding a high-fat diet (HFD), followed by intratracheal instillation of LPS to induce ALI. Pulmonary macrophages of obese mice were depleted through intratracheal instillation of clodronate liposomes. The expression of miR-192 was examined in lung tissues, primary alveolar macrophages (AMs), and the mouse alveolar macrophage cell line (MH-S) using RT-qPCR. m6A quantification and RIP assays helped determine the cause of miR-192 dysregulation. miR-192 agomir and antagomir were used to investigate its function in mice and MH-S cells. Bioinformatics and dual-luciferase reporter gene assays were used to explore the downstream targets of miR-192. In obese mice, depletion of macrophages significantly alleviated lung tissue inflammation and injury, regardless of LPS challenge. miR-192 expression in lung tissues and alveolar macrophages was diminished during obesity and further decreased with LPS stimulation. Obesity-induced overexpression of FTO decreased the m6A modification of pri-miR-192, inhibiting the generation of miR-192. In vitro, inhibition of miR-192 enhanced LPS-induced polarization of M1 macrophages and activation of the AKT/ NF-κB inflammatory pathway, while overexpression of miR-192 suppressed these reactions. BIG1 was confirmed as a target gene of miR-192, and its overexpression offset the protective effects of miR-192. In vivo, when miR-192 was overexpressed in obese mice, the activation of pulmonary macrophages and the extent of lung injury were significantly improved upon LPS challenge. Our study indicates that obesity-induced downregulation of miR-192 expression exacerbates LPS-induced ALI by promoting macrophage activation. Targeting macrophages and miR-192 may provide new therapeutic avenues for obesity-associated ALI.
Macrophage polarization by MSC-derived CXCL12 determines tumor growthSpringer Science and Business Media LLC - Tập 26 - Trang 1-15 - 2021
Shabnam Babazadeh, Seyed Mahdi Nassiri, Vahid Siavashi, Mohadeseh Sahlabadi, Mostafa Hajinasrollah, Mohamad Zamani-Ahmadmahmudi
Phenotypic and functional heterogeneity of macrophages is known to be the main reason for their ability to regulate inflammation and promote tumorigenesis. Mesenchymal stem cells (MSCs) are one of the principal cells commonly found in the tumor stromal niche, with capability of macrophage phenotypic switching. The objective of this study was to evaluate the role of C-X-C motif chemokine ligand 12 (CXCL12) produced by marrow-derived MSCs in the phenotypic and functional pattern of bone marrow-derived macrophages (BMDMs). First, the CRISPR/Cas9 system was used for the CXCL12 gene knock-out in MSCs. Then, coculture systems were used to investigate the role of MSCsCXCL12−/− and MSCsCXCL12+/+ in determination of macrophage phenotype. To further analyze the role of the MSC-derived CXCL12 niche, cocultures of 4T1 mammary tumor cells and macrophages primed with MSCsCXCL12−/− or MSCsCXCL12+/+ as well as in-vivo limiting dilution assays were performed. Our results revealed that the expression of IL-4, IL-10, TGF-β and CD206 as M2 markers was significantly increased in macrophages co-cultured with MSCsCXCL12+/+ , whereas the expression of IL-6, TNF-α and iNOS was conversely decreased. The number and size of multicellular tumor spheroids were remarkably higher when 4T1 cells were cocultured with MSCCXCL12+/+-induced M2 macrophages. We also found that the occurrence of tumors was significantly higher in coinjection of 4T1 cells with MSCCXCL12+/+-primed macrophages. Tumor initiating cells were significantly decreased after coinjection of 4T1 cells with macrophages pretreated with MSCsCXCL12−/−. In conclusion, our findings shed new light on the role of MSC-derived CXCL12 in macrophage phenotypic switching to M2, affecting their function in tumorigenesis.
Ascorbic acid inhibits the migration of walker 256 carcinosarcoma cellsSpringer Science and Business Media LLC - Tập 13 - Trang 103-111 - 2007
Ewa Wybieralska, Monika Koza, Jolanta Sroka, Jarosław Czyż, Zbigniew Madeja
The results of several experimental studies have shown that ascorbic acid inhibits tumor growth and metastasis. Ascorbic acid is an antioxidant that acts as a scavenger for a wide range of reactive oxygen species (ROS). Both tumour metastasis and cell migration have been correlated with the intracellular ROS level, so it was postulated that the inhibitory effect of ascorbic acid derivatives on cell motility may be caused by scavenging of ROS. Time-lapse analyses of Walker 256 carcinosarcoma cell migration showed that both the speed of movement and the cell displacement were inhibited by ascorbic acid applied in concentrations ranging from 10 to 250 μM. This effect correlated with a reduction in the intracellular ROS level in WC 256 cells, suggesting that ROS scavenging may be a mechanism responsible for the inhibition of WC 256 cell migration. However, another potent antioxidant, N-acetyl-L-cysteine, also efficiently decreased the intracellular ROS level in WC 256 cells, but did not affect the migration of the investigated cells. These results demonstrate that intact, unmodified ascorbic acid applied in physiologically relevant and nontoxicconcentrations exerts an inhibitory effect on the migration of WC 256 carcinosarcoma cells, and that this may be one of the factors responsible for the anti-metastatic activity of vitamin C. However, our data does not support the hypothesis that the scavenging of intracellular ROS is the main mechanism in the inhibition of cancer cell migration by ascorbic acid.
